in horses.1-3 In horses, degenerative joint disease is a ... mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chon-.
Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis Allison A. Stewart, DVM, MS; Christopher R. Byron, DVM, MS; Holly Pondenis, BS; Matthew C. Stewart, BVSc, PhD Objective—To determine whether fibroblast growth factor-2 (FGF-2) treatment of equine mesenchymal stem cells (MSCs) during monolayer expansion enhances subsequent chondrogenesis in a 3-dimensional culture system. Animals—6 healthy horses, 6 months to 5 years of age. Procedures—Bone marrow–derived MSCs were obtained from 6 horses. First-passage MSCs were seeded as monolayers at 10,000 cells/cm2 and in medium containing 0, 1, 10, or 100 ng of FGF-2/mL. After 6 days, MSCs were transferred to pellet cultures (200,000 cells/pellet) and maintained in chondrogenic medium. Pellets were collected after 15 days. Pellets were analyzed for collagen type II content by use of an ELISA, total glycosaminoglycan content by use of the dimethylmethylene blue dye–binding assay, and DNA content by use of fluorometric quantification. Semiquantitative PCR assay was performed to assess relative concentrations of collagen type II and aggrecan mRNAs. Results—Use of 100 ng of FGF-2/mL significantly increased pellet DNA and glycosaminoglycan content. Collagen type II content of the pellet was also increased by use of 10 and 100 ng of FGF-2/mL. Collagen type II and aggrecan mRNA transcripts were increased by treatment with FGF-2. Some control samples had minimal evidence of collagen type II and aggrecan transcripts after 35 cycles of amplification. Conclusions and Clinical Relevance—FGF-2 treatment of bone marrow–derived MSC monolayers enhanced subsequent chondrogenic differentiation in a 3-dimensional culture. This result is important for tissue engineering strategies dependent on MSC expansion for cartilage repair. (Am J Vet Res 2007;68:941–945)
O
steoarthritis is a major source of debilitating pain, economic cost, and reduced athletic performance in horses.1-3 In horses, degenerative joint disease is a more common problem than acute traumatic injuries or respiratory tract diseases and has a greater economic impact.2,3 Results of several studies1,3 indicate that problems involving the metacarpophalangeal, metatarsophalangeal, and carpal joints account for 25% to 28% of horses lost from training. Degenerative joint disease of the distal tarsal joints, forelimb proximal interphalangeal joints, and metacarpophalangeal joints is a common cause of reduced performance in dressage horses.4 Mesenchymal stem cells have received a great deal of attention in recent years regarding applications of their use for tissue repair and regeneration. Bone marrow–derived MSCs are capable of differentiation along osteoblastic, fibroblastic, adipogenic, and chondrogenic lineages.5 This multipotentiality makes MSCs a promising source for cell-based repair of these tissue types, in light of their excellent immunocompatability, reduced risk of disease transmisReceived November 18, 2006. Accepted March 16, 2007. From the Department of Clinical Science, College of Veterinary Medicine, University of Illinois, Urbana, IL 61802. Supported by the University of Illinois at Urbana-Champaign Campus Research Board. Address correspondence to Dr. Stewart.
MSC FGF-2 GAG TGF-b1 hMSC
Abbreviations Mesenchymal stem cell Fibroblast growth factor-2 Glycosaminoglycan Transforming growth factor b1 Adult human bone marrow–derived mes - enchymal stem cell
sion, and minimal injury to the donor site. Therapies that make use of MSCs appear to be particularly valuable for articular cartilage repair because this tissue has little intrinsic healing capacity.6 Despite the many indications for MSC use in tissue engineering, there have been difficulties associated with MSC culture and differentiation into cartilage.7–10 Mesenchymal stem cell–based models generally require a postisolation purification and expansion phase, performed in monolayer culture, to provide sufficient cells for subsequent applications. Mesenchymal stem cell expansion is followed by a differentiation phase, the conditions of which vary with the specific cell lineage required. Mesenchymal stem cells consistently have less expandability, compared with chondrocytes obtained from the same horses in identical cell culture conditions.10,11 Expanded MSCs placed in a 3-dimensional fibrin matrix also have less chondrogenic capacity, compared with chondrocytes cultured in identical conditions.10 Findings of these studies indicate the need for methods to enhance proliferation
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and subsequent chondrogenic differentiation of MSCs prior to their use for repair of articular defects. Fibroblast growth factor-2, also known as basic fibroblastic growth factor, has been used in many progenitor cell culture systems to stimulate proliferation.9,12-18 Fibroblast growth factor-2 has an important role in maintaining the self-renewal capacities of embryonic and adult progenitor cells.12,13,19 Independent of its mitogenic activities, results of several recent studies20-22 indicate that FGF-2 has specific beneficial effects on chondrogenesis and repair of cartilage defects. In those studies, exogenous FGF-2 administration significantly increased the number of MSCs recruited to an articular cartilage defect and improved the quality of the repair through production of hyaline cartilage rather than fibrous tissue.20-22 These results support a critical role for FGF-2 in MSC-based repair of articular cartilage. The purpose of the study reported here was to determine whether FGF-2 treatment of MSCs during the monolayer expansion phase increases the rate or degree of cell proliferation and enhances subsequent chondrogenesis in pelleted cell culture. Our hypothesis was that use of FGF-2 for expansion of equine bone marrow– derived MSCs would increase monolayer proliferation and subsequent chondrogenesis. Materials and Methods Cell culture—Bone marrow aspirates were obtained aseptically from the tuber coxae of 6 healthy horses of ages 6 months, 13 months, 14 months, 2 years, 2 years, and 5 years. All horses were sedated with 0.5 to 1.0 mg of xylazine/kg administered IV prior to induction of anesthesia. The horses were anesthetized with 2.2 mg of ketamine/kg and 0.1 mg of diazepam/kg, delivered via an IV catheter placed in the left jugular vein. General anesthesia was maintained via IV administration of a combination of 5% guaifenesin solution with 1,000 mg of ketamine/L and 1,000 mg of xylazine/L. The skin over the left tuber coxae was clipped and aseptically prepared, and a bone marrow biopsy needlea was used to aspirate bone marrow into 30-mL syringes containing 1,000 units of heparin diluted to a volume of 5 mL with PBS solution. Three aspirates were obtained, 1 each from 3 sites on the left tuber coxae of each horse. Aspirations were performed in < 30 minutes. Aspirates were obtained following guidelines reviewed and approved by the Institutional Animal Care and Use Committee at the University of Illinois and were obtained from horses donated to the university or with the owners’ consent prior to euthanasia. Thirty milliliters of bone marrow aspirate was diluted with 15 mL of PBS solution and centrifuged at 300 X g for 15 minutes. The supernatant was removed, and the pellet was suspended in PBS solution and centrifuged as before. The pelleted cells were suspended in 12 mL of low-glucose Dulbecco modified Eagle mediumb supplemented with 10% fetal bovine serum, 300 µg of l-glutamine/mL, 100 U of sodium penicillin/mL, 100 µg of streptomycin sulfate/mL, and 1mM sodium pyruvate/mL. The resuspended cells were placed in a 25-cm2 flask. Culture flasks were left undisturbed at 37oC in a 5% CO2 atmosphere with 90% humidity for 5 days to allow cells to attach to the culture dishes, as 942
described.10,11,23 After the initial 5 days, the medium was replaced every 2 to 3 days with 12 mL of fresh medium for 2 to 3 weeks until confluency. Cell expansion—Confluent monolayers of MSCs were trypsinized, suspended, and plated at 10,000 cells/ cm2. These first-passage cells were incubated with medium containing no FGF-2 (control) or 1, 10, or 100 ng of FGF-2c/mL. The medium was changed every 2 to 3 days, maintaining the appropriate FGF-2 concentration. After 6 days of incubation with FGF-2, the monolayers were trypsinized, counted, and transferred to pellet cultures containing 200,000 cells/pellet. Cell counts were performed as described,11 and cell viability was determined via trypan blued exclusion.24 Cell differentiation—Mesenchymal stem cells were suspended at 400,000 cells/mL of chondrogenic medium (high-glucose Dulbecco modified Eagle medium containing 5 ng of TGF-β1e/mL, 37.5 µg of ascorbic acid, 1% insulin-transferrin-selenous acid supplement, 300 µg of l-glutamine/mL, 100 U of sodium penicillin/mL, and 100 µg of streptomycin sulfate/mL). Two hundred thousand cells in 0.5 mL were placed in a conical polypropylene microcentrifuge tubef and centrifuged at 500 X g for 5 minutes to pellet the cells. Pellet cultures received chondrogenic medium every 2 to 3 days. Pellets were collected after 15 days. Six pellets were stored at –80oC for DNA and protein analysis. Three pellets were snap-frozen in liquid nitrogen for RNA isolation, and 1 pellet was placed in 4% paraformaldehyde for histologic processing. This was repeated for each of the 3 aspirates for all 6 horses in the study. Total pellet DNA content—Total DNA content of the pellets was determined via fluorometric measurement of Hoechst 33258g dye incorporation by use of reported methods.25 All samples were analyzed in duplicate, and mean values were calculated to provide a single datum point. Optical density values were converted to total DNA content per pellet by reference to a standard curve generated by serial dilutions of calf thymus DNA. Total pellet glycosoaminoglycan content—Three pellets were digested in 0.1% papainh at 65oC for 16 hours.26,27 Dimethylmethylene blue binding assays were performed to measure sulphated glycosaminoglycan concentrations and compared against a standard curve based on chondroitin sulfate.27 All samples were analyzed in duplicate, and mean values were calculated. Total pellet collagen type II content—Collagen type II protein concentrations in each pellet were determined by use of a commercially available enzyme immunosorbent assayi according to the manufacturer’s protocol. Three pellets from each treatment group were lyophilized and digested for collagen II protein quantification. All samples were compared against a standard curve based on a type II collagen standard. Samples were analyzed in duplicate, and mean values were calculated. RNA isolation, reverse transcription, and PCR amplification—Total RNA was isolated from 3 MSC pellets AJVR, Vol 68, No. 9, September 2007
from each treatment group by use of a commercially available kitj. The mRNA in each sample was converted to cDNA with a commercially available reverse transcription kitk and oligo (dT) primers. The cDNA generated from the reverse transcription reactions was then amplified via PCR with Taq DNA polymerasel and genespecific primers. Relative expression of collagen type II (Col II), aggrecan, and the reference gene elongation factor-1 alpha (EF-1α) was assessed by use of primers (Table 1). Primers were developed with software.28 Following PCR amplification, 5 µL of each sample was loaded onto a 1.5% agarose gel and electrophoresed to separate the PCR products according to size. After electrophoresis, DNA bands in the gels were stained with ethidium bromide and viewed under UV light. Digital images of the gels were captured by use of gel imaging software,m and relative band intensities were quantified by use of imaging software.n Collagen type II and aggrecan gel band intensity levels were normalized against EF-1α gel band intensity levels.
use of the Holm-Sidak method were used to evaluate mean differences in response for significant effects. A value of P ≤ 0.05 was considered significant.
Histologic examination—After 24 hours of fixation in 4% paraformaldehyde, the pellets were transferred to PBS solution and stored at 4oC. The pellets were dehydrated in alcohol, embedded in paraffin, sectioned, and stained with toluidine blue.
Total pellet GAG content—Treatment of MSC monolayers with 100 ng of FGF-2/mL caused a 2.5times increase (P < 0.001) in total GAG concentration of the pellets, compared with control cultures. Treatment with 1 and 10 ng/mL did not significantly increase total pellet GAG concentration. When total GAG content was normalized for DNA, treatment with FGF-2 was no longer significant.
Statistical analysis—Mean values for monolayerexpanded cell numbers, pellet GAG concentration, pellet collagen type II concentration, pellet DNA concentration, normalized aggrecan mRNA concentrations, and normalized collagen type II mRNA concentrations were calculated for each horse in the study. The effect of FGF-2 on these values was evaluated by use of a 2-way ANOVAo with horse as a covariate to control for withinsubject effects. Pairwise multiple comparisons made by
Results Cell counts after monolayer expansion phase— There was considerable variation in monolayer expansion in response to FGF-2 treatment. Some MSC samples had increased monolayer cell numbers with FGF-2 treatment, whereas other MSC samples had minimal response. Treatment with FGF-2 had no significant (P = 0.406) effect on first-passage MSC numbers obtained after 6 days of expansion in monolayer. Total pellet DNA content—Addition of FGF-2 during monolayer expansion increased the DNA content of MSC pellets 2.7 times after 15 days in chondrogenic medium at the 100 ng/mL dose (P < 0.001), compared with untreated MSC pellet controls. No significant effect was detected with lower FGF-2 concentrations.
Total pellet collagen II content—Both 10 and 100 ng/mL concentrations of FGF-2 significantly (P = 0.006) increased collagen type II concentration. In contrast to total DNA and GAG concentrations, the effect on collagen type II deposition was not dose dependent. Treatment of MSC monolayers with 10 and
Table 1—Primers used for PCR ampliflication of genes in a study of FGF-2 and equine MSCs. Gene
Sequence
Cycle
Amplicon (bp)
Collagen type II
Sense 5’-AGC AGG AAT TTG GTG TGG AC-3’ Antisense 3’-TCT GCC CAG TTC AGG TCT CT-5’
62˚C, 30 s 24 or 27 cycles
223
Aggrecan
Sense 5’-GAC GCC GAG AGC AGG TGT-3’ Antisense 3’- AAG AAG TTG TCG GGC TGG TT-5’
62˚C, 30 s 24 or 27 cycles
202
EF-1α
Sense 5’-CCC GGA CAC AGA GAC TTC AT-3’ Antisense 3’-AGC ATG TTG TCA CCA TTC CA-5’
62˚C, 30 s 21 or 24 cycles
328
Figure 1—Photomicrographic views of histologic sections of equine MSC pellets treated with various concentrations of FGF-2. Toluidine blue stain; internal scale marker applies to all 4 photomicrographs. AJVR, Vol 68, No. 9, September 2007
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100 ng of FGF-2/mL resulted in almost identical (4.0times) increases in collagen type II protein concentration in pellets, compared with control cultures. When the collagen II protein values were normalized for total DNA concentration of the pellet, there was no significant difference with FGF-2 treatment. Interestingly in this study, with limited horse numbers, there was no measurable association between donor age and monolayer expansion or any of the subsequent chondrogenic outcomes that were measured. Aggrecan and collagen type II mRNAs—After normalization with EF-1α, aggrecan and collagen type II expression levels indicated a 1.4- to 2.7-times increase (P < 0.001) in aggrecan and a 1.3- to 1.4-times increase (P = 0.01) in collagen type II transcripts with FGF-2 treatment. These results were consistent with the GAG and collagen type II data. The total RNA yields from these experiments were low. Therefore, reverse transcription was carried out on the total RNA sample obtained from 3 pellets combined from each treatment group. There were few or no detectable collagen type II and aggrecan transcripts in some of the control group samples after 35 cycles of amplification, suggesting minimal chondrogenesis occurred in MSC pellets that were not pretreated with FGF-2 during monolayer expansion. Histologic findings—Pellets treated with 10 and 100 ng of FGF-2/mL were larger in size than controls and had more intense toluidine blue staining of proteoglycans, consistent with the total pellet GAG concentration, total pellet DNA concentration, and reverse transcriptase–PCR data (Figure 1). Discussion Results of this study indicated that FGF-2 administered during equine MSC monolayer expansion did not enhance cell numbers. However, FGF-2 administration during MSC monolayer expansion had significant effects on subsequent MSC chondrogenesis during the pellet phase of this model. Use of FGF-2 during monolayer expansion increased pellet DNA concentration, resulting in subsequent increases in collagen type II and total GAG concentrations. These results were similar to a recent study9 that used FGF-2 to expand adult human bone marrow–derived cells, in which chondrogenic cultures derived from FGF-treated cells were larger and contained more proteoglycan than those made from control cells. The histologic results of the present study were also consistent with that study9; FGF-expanded cells formed a more uniform matrix distributed through the entire pellet. Both studies revealed a peripheral shell of fibrous-like tissue in the untreated controls that disappeared with FGF-2 administration. There was considerable variation in the response of MSCs to addition of FGF-2, in monolayer expansion and chondrogenesis. Some studies29,30 reveal an association between donor age and the quality and responsiveness of bone marrow–derived MSCs. However, in the present study, with limited horses, there was no measurable association between donor age and monolayer expansion or subsequent chondrogenesis. Some MSCs increased in monolayer cell numbers with FGF-2 944
treatment, whereas others had minimal response. There was no significant effect of FGF-2 on the proliferative activity of the monolayer cultures. Adult human bone marrow–derived MSCs also have variable responses of 2 kinds.9 Some hMSCs have dose-dependent mitotic responses to FGF-2, whereas others have a threshold effect.9 In a recent study,9 hMSCs that were expanded with FGF-2 replicated 3 times as fast as hMSCs under control conditions. Many studies reveal a marked difference in the mitogenic capacity and chondrogenic potential of MSCs, depending on cell densities and various culture conditions. Most researchers acknowledge the importance of testing different serum batches for low endotoxin and the ability to support MSC monolayer expansion.8,9,16 The lack of mitogenic response to FGF-2 in the present study may have been caused in part by addition of serum. The study used the same lot number of serum with low endotoxin that had been tested for equine MSC monolayer expansion. It is possible that addition of serum (10%) maximized the mitogenic capacity of the MSC monolayer cultures, masking any further mitogenic effect that could be attained by further addition of FGF-2. In contrast to other studies,9,26 in the present study, 5 ng of TGF-β1/mL was added and dexamethasone was not used for study of 3-dimensional chondrogenic differentiation; the dose of TGF-β1 was determined on the basis of a previous optimization study.11 In the present study, supplementation of equine MSC monolayers with 100 ng of FGF-2/mL significantly increased the total pellet DNA, collagen type II, and GAG content. However, when the pellet matrix protein data were normalized for DNA content, significant differences among FGF-2 dose groups were not present. This suggested that the primary effect of FGF-2 expansion was to increase pellet matrix protein through an increase in DNA content. This increase in DNA content may have occurred by increasing or retaining the cell number in each pellet. These results contrast with a recent study9 in which hMSCs expanded with FGF-2 did not develop a significant increase in pellet DNA content when transferred to 3-dimensional pellet cultures. The difference between these 2 studies may be attributed to differences in species or differences in supplementation of the 3-dimensional culture system. The present study did not use dexamethasone supplementation that may have increased the DNA content of the control cultures. In addition, the difference in TGF-β1 supplementation between the 2 studies9 (5 vs 10 ng/mL) may have altered the effect of FGF-2 on the DNA content of the pellets. The increase in pellet cell number in the present study may have resulted from enhanced cell proliferation during the differentiation phase after addition of FGF2. Alternately, it could have resulted from enhanced cell survival after the trypsinization and pelleting phase of the experiment. Although sequence homology for FGF-2 is very high (> 90%) across a wide range of species, there are many isoforms and splicing variants.31,32 Fibroblast growth factor-2 has at least 5 isoforms, and its biological functions are probably isoform specific.31,32 In vivo, the 18-kd heparin-binding protein is produced as a 155-amino acid precursor that matures to a 146-amino AJVR, Vol 68, No. 9, September 2007
acid residue.32 Human recombinant forms of FGF-2 are available as 146 and 153 amino acids. These recombinant forms may have different biological activities. In the present study, the 153-amino acid human recombinant FGF-2 was used. Many studies9,12-22 reveal the importance of FGF-2 supplementation in the maintenance of progenitor cell populations. The present study revealed beneficial effects of the 100 ng/mL concentration to expand equine bone marrow–derived progenitor cells for subsequent chondrogenesis. The beneficial effects of FGF-2 on cell numbers and extracellular matrix deposition will be valuable for accomplishing future tissue engineering goals. a.
Jamshidi bone marrow biopsy needle, Cardinal Health, Dublin, Ohio. b. Dulbecco modified Eagle medium, Mediatech Inc, Herndon, Va. c. Fibroblast growth factor-2, Sigma Chemical Co, St Louis, Mo. d. Trypan blue, Sigma Chemical Co, St Louis, Mo. e. Transforming growth factor-β1, R & D Systems, Minneapolis, Minn. f. Polypropylene conical tube, BD Biosciences, Franklin Lakes, NJ. g. Hoechst 33258, Sigma Chemical Co, St Louis, Mo. h. Papain, Sigma Chemical Co, St Louis, Mo. i. Native type II collagen detection kit, Chondrex Inc, Redmond, Wash. j. RNeasy mini kit, Qiagen, Valencia, Calif. k. Superscript II, Invitrogen, Carlsbad, Calif. l. Platinum Taq DNA polymerase, Invitrogen, Carlsbad, Calif. m. Scientific Imaging Systems, Rochester, NY. n. Image J software, Research Services Branch, National Institutes of Health, Bethesda, Md. o. SigmaStat, Systat Software Inc, San Jose, Calif.
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